The complement system is composed of many different proteins that are important in the immune system's response to foreign antigens. The proteins of the complement system constitute about 10% of the globulins in normal serum. The complement system becomes activated when its primary components are cleaved, and the resulting products, either alone or with other proteins, activate additional complement proteins, resulting in a proteolytic cascade. Activation of the complement system leads to a variety of responses including increased vascular permeability, chemotaxis of phagocytic cells, activation of inflammatory cells, opsonization of foreign particles, direct killing of cells and tissue damage. In the classical pathway, activation of the complement system is triggered by antigen-antibody complexes. In an alternative pathway, the complement system may be catalyzed, for example, by lipopolysaccharides present in cell walls of pathogenic bacteria.
Complement receptor type I (CR1) is a membrane glycoprotein that has been shown to be present on erythrocytes (E), monocytes/macrophages, granulocytes, B cells, some T cells, splenic follicular dendritic cells, and glomerular podocytes. CR1 mediates the binding of particles or immune complexes that contain activated complement to the surface of these cells. More specifically, CR1 binds to the complement activation products C3b and C4b, and has thus also been referred to as the C3b/C4b receptor. The consequences of these interactions depend upon the cell type bearing the receptor (Fearon, D. T. and Wong, W. W. (1983) Ann. Rev. Immunol. 1:243). CR1 on the surface of erythrocytes binds immune complexes for transport to the liver (Cornacoff, J. B. et al. (1983) J. Clin. Invest. 71:236; Nedof, N. E. et al. (1982) J. Exp. Med. 145:1739). CR1 on neutrophils and monocytes internalizes bound complexes, either by adsorptive endocytosis through coated pits or by phagocytosis after activation of the receptor by phorbol esters, chemotactic peptides or proteins that are present in the extracellular matrix, such as fibronectin and laminin (Newman S. L. et al. (1980) J. Immunol. 124:2236; Wright, S. D. and Silverstein S. C. (1982) J. Exp. Med. 145:1149; Wright S. D. et al. (1983) J. Exp. Med. 148:1338). Phosphorylation of CR1 may have a role in the acquisition of this phagocytic activity (Changelian P. S. & Fearon D. T. (1986) J. Exp. Med. 163:101). The function of CR1 on B lymphocytes is less defined, although treatment of these cells with antibody to CR1 enhanced their response to suboptimal doses of pokeweed mitogen (Daha, M. R. et al. (1983) Immunobiol. 164:227 (Abstr)). CR1 on follicular dendritic cells may serve an antigen presentation role (Klaus G. G. B. et al. (1980) Immunol. Rev. 53:3).
In addition to serving as a receptor, CR1 also has complement regulatory functions. For example, in the classical and alternative complement pathways, CR1 can inhibit the C3/C5 convertases and can also act as a cofactor for the cleavage of C3b and C4b by factor I (Fearon D. T. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:5867; Liida X. & Nussenzweig V. (1981) J. Exp. Med. 153:1138). In the classical pathway of complement activation, the complex C4b, 2a is a C3 activating enzyme, or convertase. CR1 can bind to C4b and promote the dissociation of C4b,2a. This binding renders C4b susceptible to irreversible proteolytic inactivation by factor I through cleavage to the inactivated complement proteins C4c and C4d. In the alternative pathway of complement activation, the complex C3b, 4b is a C3 convertase. CR1 can bind to C3b and can also promote the dissociation of C3b, 4b. Formation of C3b, CR1 renders C3b susceptible to irreversible proteolytic inactivation by factor I, resulting in the formation of the inactivated complement protein C3b (iC3b).
CR1 is a glycoprotein composed of a single polypeptide chain. Four allotypic forms of CR1 have been found, differing in molecular weight by increments of ˜40,000-50,000 daltons. The two most common forms are the F and S allotypes (also termed the A and B allotypes), having molecular weights of 250,000 and 290,000 daltons (Dykman, T. R. et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 30:1698; Wong, W. W. et al. (1983) J. Clin. Invest. 72:685), respectively. There are also two rarer forms having molecular weights of 210,000 and >290,000 daltons (Dykman, T. R. et al. (1984) J. Exp. Med. 159:691; Dykman, R. R. et al. (1985) J. Immunol. 134:1787). These differences apparently represent variations in the polypeptide chain of CR1, rather than glycosylation state (Wong W. W. et al. (1983) J. Clin. Invest. 72:685). All four CR1 allotypes have C3b binding activity (Dykman, T. R. et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 30:1698; Dykman, T. R. et al. (1984) J. Exp. Med. 159:691; Dykman, T. R. et al. (1985) J. Immunol. 134:1787; Wong W. W. et al (1983) J. Clin. Invest. 72:685).
While CR1 is found on various hematopoietic cells, the vast majority of CR1 in the blood is present specifically on erythrocytes. This CR1 plays a predominant role in the processing and clearance of circulating immune complexes. Once a pathogen adheres to the erythrocyte, the immune-complexed pathogen is efficiently transferred to acceptor phagocytic cells, such as fixed tissue macrophages in the liver and spleen. Interestingly, the pathogen in this transfer reaction is apparently stripped from the erythrocyte without any discernible damage to the erythrocyte (Lindorfer M. A. et al. (2001) Immun. Reviews 183:10-24). The detailed mechanism of the transfer reaction has not yet been fully elucidated. Studies suggest, however, that the in vivo transfer reaction is facilitated by a process that is unlikely to depend upon Factor I-mediated release (Lindorfer et al. (2001) Immun. Reviews 183:10-24).
An important goal in the pharmaceutical industry is the development of novel therapeutic modalities that can target and clear pathogens from tissues or the bloodstream. Several approaches undertaken to address this problem are aimed at taking advantage of the immune adherence function of primate erythrocytes. In one approach, bispecific monoclonal antibodies complexes (heteropolymers, HP) are designed to bind and immobilize a target pathogen to CR1 of a primate erythrocyte (Lindorfer M. A. et al. (2001) Immun. Reviews 183:10-24). For example, heteropolymers can consist of a monoclonal antibody specific for CR1 that is chemically cross-linked with a monoclonal antibody specific for the target pathogen. In this way, the antibody specific for CR1 serves as a surrogate for the natural CR1 ligand, C3b. One advantage of this approach is that while the natural affinity of C3b for CR1 is low, thus requiring robust complement activation and the capture of multiple C3b molecules to insure erythrocyte binding, several mouse monoclonal antibodies have been raised that possess very high affinity for CR1. Crosslinking these antibodies to a high-affinity pathogen-specific monoclonal antibody should allow for virtually any target pathogen to be bound by erythrocytes in the absence of complement. This strategy has been successfully applied to a number of bacteria and viruses (Powers J. H. et al. (1995) Infect. Immun 63:1329-1335; Kuhn S. E. et al. (1998) J. Immunol. 160:5088-5097; Nardin A., et al. (1998) 211:21-31; Hahn C. S. et al. (2001) J. Immunol 166:1057-1065).
In a related approach, the use of erythrocytes and bispecific reagents to target molecules in the serum and/or circulation can be extended to target the numerous autoantibodies associated with autoimmune diseases, such as the IgG anti-dsDNA antibodies in systemic lupus erythematosus (SLE). The autoantibodies can be targeted for erythrocyte-mediated clearance by using antigen-based heteropolymers (AHP) (Lindorfer M. A. et al. (2001) Immun. Reviews 183:10-24). Antigen-based heteropolymers consist of an autoantigen chemically cross-linked to an anti-CR1 monoclonal antibody. The erythrocyte-bound antigen-based heteropolymer captures autoantibodies and directs the newly formed erythroycte-bound immune complex into the transfer reaction for ultimate clearance to the liver.
There is a clear need in the art for animal models that can be used to test the ability of bispecific compositions, such as heteropolymers which bind CR1, to clear targeted molecules from tissues, serum and/or the circulation. Such animal models would be useful in evaluating both the efficacy and safety of bispecific compositions and bispecific composition-based therapies. Unfortunately, while mice are an excellent model for drug testing, mice do not express CR1 on their erythrocytes (Kalli and Fearon. 1994. J. Immunol. 152:2894).